Expression of miR-15/107 Family MicroRNAs in Human Tissues and Cultured Rat Brain Cells

The miR-15/107 family comprises a group of 10 paralogous microRNAs (miRNAs), sharing a 5\u27 AGCAGC sequence. These miRNAs have overlapping targets. In order to characterize the expression of miR-15/107 family miRNAs, we employed customized TaqMan Low-Density micro-fluid PCR-array to investigate the expression of miR-15/107 family members, and other selected miRNAs, in 11 human tissues obtained at autopsy including the cerebral cortex, frontal cortex, primary visual cortex, thalamus, heart, lung, liver, kidney, spleen, stomach and skeletal muscle. miR-103, miR-195 and miR-497 were expressed at similar levels across various tissues, whereas miR-107 is enriched in brain samples. We also examined the expression patterns of evolutionarily conserved miR-15/107 miRNAs in three distinct primary rat brain cell preparations (enriched for cortical neurons, astrocytes and microglia, respectively). In primary cultures of rat brain cells, several members of the miR-15/107 family are enriched in neurons compared to other cell types in the central nervous system (CNS). In addition to mature miRNAs, we also examined the expression of precursors (pri-miRNAs). Our data suggested a generally poor correlation between the expression of mature miRNAs and their precursors. In summary, we provide a detailed study of the tissue and cell type-specific expression profile of this highly expressed and phylogenetically conserved family of miRNA genes

A Customized Quantitative PCR MicroRNA Panel Provides a Technically Robust Context for Studying Neurodegenerative Disease Biomarkers and Indicates a High Correlation Between Cerebrospinal Fluid and Choroid Plexus MicroRNA Expression

MicroRNA (miRNA) expression varies in association with different tissue types and in diseases. Having been found in body fluids including blood and cerebrospinal fluid (CSF), miRNAs constitute potential biomarkers. CSF miRNAs have been proposed as biomarkers for neurodegenerative diseases; however, there is a lack of consensus about the best candidate miRNA biomarkers and there has been variability in results from different research centers, perhaps due to technical factors. Here, we sought to optimize technical parameters for CSF miRNA studies. We examined different RNA isolation methods and performed miRNA expression profiling with TaqMan® miRNA Arrays. More specifically, we developed a customized CSF-miRNA low-density array (TLDA) panel that contains 47 targets: miRNAs shown previously to be relevant to neurodegenerative disease, miRNAs that are abundant in CSF, data normalizers, and controls for potential blood and tissue contamination. The advantages of using this CSF-miRNA TLDA panel include specificity, sensitivity, fast processing and data analysis, and cost effectiveness. We optimized technical parameters for this assay. Further, the TLDA panel can be tailored to other specific purposes. We tested whether the profile of miRNAs in the CSF resembled miRNAs isolated from brain tissue (hippocampus or cerebellum), blood, or the choroid plexus. We found that the CSF miRNA expression profile most closely resembles that of choroid plexus tissue, underscoring the potential importance of choroid plexus-derived signaling through CSF miRNAs. In summary, the TLDA miRNA array panel will enable evaluation and discovery of CSF miRNA biomarkers and can potentially be utilized in clinical diagnosis and disease stage monitoring

Novel Human \u3cem\u3eABCC9/SUR2\u3c/em\u3e Brain-Expressed Transcripts and an eQTL Relevant to Hippocampal Sclerosis of Aging

ABCC9 genetic polymorphisms are associated with increased risk for various human diseases including hippocampal sclerosis of aging. The main goals of this study were 1 \u3e to detect the ABCC9 variants and define the specific 3′ untranslated region (3′UTR) for each variant in human brain, and 2 \u3e to determine whether a polymorphism (rs704180) associated with risk for hippocampal sclerosis of aging pathology is also associated with variation in ABCC9 transcript expression and/or splicing. Rapid amplification of ABCC9 cDNA ends (3′RACE) provided evidence of novel 3′ UTR portions of ABCC9 in human brain. In silico and experimental studies were performed focusing on the single nucleotide polymorphism, rs704180. Analyses from multiple databases, focusing on rs704180 only, indicated that this risk allele is a local expression quantitative trait locus (eQTL). Analyses of RNA from human brains showed increased ABCC9 transcript levels in individuals with the risk genotype, corresponding with enrichment for a shorter 3′ UTR which may be more stable than variants with the longer 3′ UTR. MicroRNA transfection experiments yielded results compatible with the hypothesis that miR‐30c causes down‐regulation of SUR2 transcripts with the longer 3′ UTR. Thus we report evidence of complex ABCC9 genetic regulation in brain, which may be of direct relevance to human disease

MicroRNA Expression Patterns in Human Anterior Cingulate and Motor Cortex: A Study of Dementia with Lewy Bodies Cases and Controls

Overview MicroRNAs (miRNAs) have been implicated in neurodegenerative diseases including Parkinson’s disease and Alzheimer’s disease (AD). Here, we evaluated the expression of miRNAs in anterior cingulate (AC; Brodmann area [BA] 24) and primary motor (MO; BA 4) cortical tissue from aged human brains in the University of Kentucky AD Center autopsy cohort, with a focus on dementia with Lewy bodies (DLB). Methods RNA was isolated from gray matter of brain samples with pathology-defined DLB, AD, AD+DLB, and low-pathology controls, with n=52 cases initially included (n=23 with DLB), all with low (\u3c4hrs) postmortem intervals. RNA was profiled using Exiqon miRNA microarrays. Quantitative PCR for post-hoc replication was performed on separate cases (n=6 controls) and included RNA isolated from gray matter of MO, AC, primary somatosensory (BA 3), and dorsolateral prefrontal (BA 9) cortical regions. Results The miRNA expression patterns differed substantially according to anatomic location: of the relatively highly-expressed miRNAs, 150/481 (31%) showed expression that was different between AC versus MO (at p\u3c0.05 following correction for multiple comparisons), most (79%) with higher expression in MO. A subset of these results were confirmed in qPCR validation focusing on miR-7, miR-153, miR-133b, miR-137, and miR-34a. No significant variation in miRNA expression was detected in association with either neuropathology or sex after correction for multiple comparisons. Conclusion A subset of miRNAs (some previously associated with α-synucleinopathy and/or directly targeting α-synuclein mRNA) were differentially expressed in AC and MO, which may help explain why these brain regions show differences in vulnerability to Lewy body pathology

Mitochondria-Associated MicroRNAs in Rat Hippocampus Following Traumatic Brain Injury

Traumatic brain injury (TBI) is a major cause of death and disability. However, the molecular events contributing to the pathogenesis are not well understood. Mitochondria serve as the powerhouse of cells, respond to cellular demands and stressors, and play an essential role in cell signaling, differentiation, and survival. There is clear evidence of compromised mitochondrial function following TBI; however, the underlying mechanisms and consequences are not clear. MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate gene expression post-transcriptionally, and function as important mediators of neuronal development, synaptic plasticity, and neurodegeneration. Several miRNAs show altered expression following TBI; however, the relevance of mitochondria in these pathways is unknown. Here, we present evidence supporting the association of miRNA with hippocampal mitochondria, as well as changes in mitochondria-associated miRNA expression following a controlled cortical impact (CCI) injury in rats. Specifically, we found that the miRNA processing proteins Argonaute (AGO) and Dicer are present in mitochondria fractions from uninjured rat hippocampus, and immunoprecipitation of AGO associated miRNA from mitochondria suggests the presence of functional RNA-induced silencing complexes. Interestingly, RT-qPCR miRNA array studies revealed that a subset of miRNA is enriched in mitochondria relative to cytoplasm. At 12h following CCI, several miRNAs are significantly altered in hippocampal mitochondria and cytoplasm. In addition, levels of miR-155 and miR-223, both of which play a role in inflammatory processes, are significantly elevated in both cytoplasm and mitochondria. We propose that mitochondria-associated miRNAs may play an important role in regulating the response to TBI

The miR-15/107 Family of microRNA Genes Regulates CDK5R1/p35 with Implications for Alzheimer’s Disease Pathogenesis

Cyclin-dependent kinase 5 regulatory subunit 1 (CDK5R1) encodes p35, the main activatory subunit of cyclin-dependent kinase 5 (CDK5). The p35/CDK5 active complex plays a fundamental role in brain development and functioning, but its deregulated activity has also been implicated in various neurodegenerative disorders, including Alzheimer\u2019s disease (AD). CDK5R1 displays a large and highly evolutionarily conserved 3\u2032-untranslated region (3\u2032-UTR), a fact that has suggested a role for this region in the post-transcriptional control of CDK5R1 expression. Our group has recently demonstrated that two miRNAs, miR-103 and miR-107, regulate CDK5R1 expression and affect the levels of p35. MiR-103 and miR-107 belong to the miR-15/107 family, a group of evolutionarily conserved miRNAs highly expressed in human cerebral cortex. In this work, we tested the hypothesis that other members of this group of miRNAs, in addition to miR-103 and miR-107, were able to modulate CDK5R1 expression. We provide evidence that several miRNAs belonging to the miR-15/107 family regulate p35 levels. BACE1 expression levels were also found to be modulated by different members of this family. Furthermore, overexpression of these miRNAs led to reduced APP phosphorylation levels at the CDK5-specific Thr668 residue. We also show that miR-15/107 miRNAs display reduced expression levels in hippocampus and temporal cortex, but not in cerebellum, of AD brains. Moreover, increased CDK5R1 mRNA levels were observed in AD hippocampus tissues. Our results suggest that the downregulation of the miR-15/107 family might have a role in the pathogenesis of AD by increasing the levels of CDK5R1/p35 and consequently enhancing CDK5 activity

IDENTIFICATION OF HYPOXIA-REGULATED MICRORNAs IN MATERNAL BLOOD AS EARLY PERIPHERAL BIOMARKERS FOR FETAL GROWTH RESTRICTION

Current tests available to diagnose fetal hypoxia in-utero lack sensitivity thus failing to identify many fetuses at risk. Emerging evidence suggests that microRNAs derived from the placenta circulate in the maternal blood during pregnancy and may be used as non-invasive biomarkers for pregnancy complications. With the intent to identify putative markers of fetal growth restriction(FGR) and new therapeutic druggable targets, we examined, in maternal blood samples, the expression of a group of microRNAs, known to be regulated by hypoxia. The expression of microRNAs was evaluated in maternal plasma samples collected from (1) women carrying a preterm FGR fetus(FGR group) or (2) women with an appropriately grown fetus matched at the same gestational age (Control group). To discriminate between early- and late-onset FGR, the study population was divided into two subgroups according to the gestational age at delivery. Four microRNAs were identified as possible candidate for the diagnosis of FGR: miR-16-5p, miR-103-3p, miR-107-3p, and miR-27b-3p that were upregulated in FGR blood samples before the 32nd week of gestation as compared to aged matched control group. By contrast, miRNA103-3p and miRNA107-3p analyzed between the 32nd and 37th week of gestation showed lower expression in the FGR group compared to aged matched controls. Notably, the expression of all miRNAs was increased through gestation in healthy control group. Our results showed that measurement of miRNAs in maternal blood may form the basis for a future diagnostic test to determine the degree of fetal hypoxia in FGR, thus allowing the start of appropriate therapeutic strategies in order to alleviate the burden of this disease

Metabolic rewiring of the failing heart:unravelling cause and effect

Heart failure (HF) currently affects more than 37 million people globally and is rising in prevalence. The 50% of HF patients who suffer from heart failure with preserved ejection fraction (HFpEF) also commonly present with accompanying diseases associated with the metabolic syndrome, such as obesity or type II diabetes. Changes in the cardiac fuel handling, or metabolism, are increasingly recognized as important in the development of HFpEF. However, their exact contribution remains unclear and therefore successful treatment strategies are lacking. This thesis expands the current knowledge of HF pathophysiology, in particular the contribution of cardiometabolic alterations in HFpEF development. The findings demonstrate that the cardiac metabolism is altered considerably in HFpEF associated with the metabolic syndrome, particularly affecting the mitochondrial metabolism within the heart. Moreover, modulating the metabolism of different cell types present in the heart can alter their functional characteristics, supporting the notion that cardiac metabolism is a target for HFpEF therapy

MiRNA influences in mesenchymal stem cell commitment to neuroblast lineage development

A thesis submitted in partial fulfilment of the requirements of the University of Wolverhampton for the degree of Professional Doctorate in Biomedical Science.Mesenchymal Stem Cells (MSCs) are multipotent stromal cells that can self-renew and differentiate into cells from the mesodermal cell lineage. Under specific conditions, MSCs are known to transdifferentiate into the neuronal cell lineage. The aim of this study was to investigate if specific microRNAs (miRNAs) are responsible for neural induction, differentiation and fate specification and if they can induce MSCs to commit to a neuroblast and/or mature neuron cell lineage. The selected miRNAs were miR-107, miR-124 and miR-381, which respectively are known to promote neurogenesis, neural differentiation and neural proliferation. Targeting protein expression by transiently destroying the messenger RNA using miRNAs is an alternative to destroying the gene permanently and, being a transient process, the cells being differentiated should not retain any permanent evidence of this process. The objectives of this study were to culture MSCs from the Wharton’s Jelly, transform these MSCs into neural-like cells using the spent medium from the neuroblastoma cell line SH-SY5Y, and further treat these conditioned cells with retinoic acid to induce further maturation. MSCs were characterised by trilineage differentiation and all three cell types were tested for a series of CD and neural markers. Once characterised, the three cell types were transfected with one of the three selected miRNAs and a target gene for each miRNA was analysed but results were sub-optimal. The MSCs and neural-like derivatives were then tested for a selection of neural markers. Once again results provided limited information, however it was observed that miR-107 and miR-124 have the potential to induce MSCs to differentiate into neural-like cells and that these miRNAs may also induce intermediate neural progenitors and immature neuron cell types to differentiate further.Malta Government Scholarship Scheme